1. Field of the Invention
The present invention relates to peptides and related compounds and in particular to peptides and related compounds which affect cell migration.
2. Description of the Related Art
Fibronectin is a widely distributed glycoprotein present at high concentrations in most extracellular matrices, in plasma (300 μg/ml), and in other body fluids. Fibronectin is a prominent adhesive protein and mediates various aspects of cellular interactions with extracellular matrices including migration. Its principal functions appear to be in cellular migration during development and wound healing, regulation of cell growth and differentiation, and haemostasis/thrombosis.
Fibronectin is a dimer of two non-identical subunits covalently linked near their COOH-termini by a pair of disulphide bonds. The difference between the subunits is determined by alternative splicing of the IIICS (or V) region. In the insoluble, matrix form of fibronectin, the dimer associates into disulphide-bonded oligomers and fibrils, while soluble, body fluid fibronectin is predominantly dimeric. Three regions of fibronectin are subject to alternative splicing and in general the matrix form of the molecule has a higher content of these segments than the soluble form. The human IIICS region has five potential variations, while the rat, bovine and chicken sequences have three, three and two, respectively. Each subunit is composed of a series of structurally independent domains linked by flexible polypeptide segments. At the primary sequence level, the origin of the majority of the fibronectin molecule can be accounted for by endoduplication of three types of polypeptide repeat. Different fibronectin domains are specialized for binding extracellular matrix macromolecules or bacterial or eukaryotic membrane receptors. The central cell-binding domain is recognised by most adherent cells via the integrin receptors α3β1, α5β1, αVβ1, αllbβ3, αVβ3, αVβ5 and αVβ6. The IIICS/Hepll cell-binding domain is recognised by lymphoid cells, neural crest derivatives and myoblasts via the integrins α4β1 and α4β7. Several peptide active sites have been identified in these domains.
Plasma fibronectin can be purified by a combination of gelatin and heparin affinity chromatography. Cell-associated fibronectin can be extracted from culture monolayers with 1 M urea. Further details on fibronectin are in The Extracellular Matrix Facts Book, Ayad et al (eds), Academic Press, Harcourt Brace & Company, London.
Limited proteolytic digestion of fibronectin results in the release of a number of its functional domains, which are characterised by their specific adhesion to other matrix macromolecules or integrin receptors on the cell surface (i.e. the cell-binding domain)1. The transmembrane assay has commonly been used to study the effects of fibronectin and its purified functional domains on cell migration in vitro. Essentially, this assay involves assessing cell movement through a polycarbonate membrane coated with an adhesive protein (usually gelatin) separating upper and lower medium compartments containing the putative effector molecule. Previous studies have revealed that nano- to micromolar concentrations of fibronectin and its purified cell-binding domain stimulate the migration of a wide range of cell types in the transmembrane assay2,3. Related studies implicated the RGD (SEQ ID NO 4) amino acid motif (located in the tenth type III repeat module) in mediating these effects of both native fibronectin and its cell-binding domain3. Significantly, small RGD-containing synthetic peptides did not stimulate cell migration; indeed, these peptides inhibited the adhesive and migration stimulating activity of larger protein domains containing the RGD (SEQ ID NO 4) motif by competition for receptor ligation4. In contrast to the activity of the cell-binding domain, the gelatin-binding domain of fibronectin has consistently been reported to be devoid of migration stimulating activity in the transmembrane assay2,5.
Schor et al (1994) Progress in Growth Factor Research 5, 223–248 is a review of cytokine control of cell motility and its modulation and mediation by the extracellular matrix. Schor et al (1993) In: Cell Behaviour: Adhesion and Motility (ed. G. Evans, C. Wigley and R. Warn) Society of Experimental Biology Symposium No. 47, pages 235–251 relates to migration stimulating factor (MSF).
Grey et al (1989) Proc. Natl. Acad. Sci. USA 86, 2438–2442 relates to the purification of the MSF produced by fetal and breast cancer patient fibroblasts.
U.S. Pat. No. 5,300,630 relates to oncodevelopmentally regulated antigens related to fibronectin.
U.S. Pat. No. 5,510,328 relates to methods of reducing or inhibiting wound contraction using certain peptides.
U.S. Pat. No. 5,354,736 relates to compounds that have enhanced cell binding with respect to collagen.
U.S. Pat. No. 4,976,734 relates to a method of stimulating chemotaxis towards a prosthetic device.
U.S. Pat. No. 4,980,279 relates to portions of fibronectin.
U.S. Pat. No. 5,049,658 relates to a polypeptide having the cell-spreading activity of human fibronectin.
U.S. Pat. No. 5,124,155 relates to wound healing dressings, which are prepared by flocculating fibronectin.
U.S. Pat. No. 5,453,489 relates to polypeptides, which encompass fibronectin—fibronectin binding sites and which are capable of inhibiting fibronectin matrix assembly.
U.S. Pat. No. 5,192,746 relates to compounds having the property of modulating cell adhesion.
U.S. Pat. No. 5,491,130 relates to peptides derived from human endothelial cell thrombosponding, which bind to the gelatin-binding domain of fibronectin.